Recent advances in pulsed power technology culminated in engineering of new devices capable of delivering high-voltage, nanosecond-duration electric pulses (nsEP) to low-impedance loads such as biological tissues and cell samples. We found that nsEP can be employed as a unique tool to modify physiology of the plasma membrane in living cells and alter cell function. The most remarkable effect of nsEP was opening of long-lived, voltage- and current-sensitive, rectifying, ion-selective, asymmetrical pores of nano- or sub- nanometer diameter (nanopores). These complex behaviors are normally expected only from sophisticated devices like protein ion channels and distinguish nanopores from conventional (larger) electropores. Once induced, nanopores oscillated between open and quasi-open (electrically silent) states for minutes, followed by either gradual resealing or abrupt breakdown into larger pores, with immediate loss of nanopore-specific properties. Nanopores appeared adequately equipped for certain functions that are traditionally ascribed to classic ion channels; we hypothesize that nanopores may form under physiological and pathological conditions to supplement ion channels as an additional ion transport pathway. Nanopores have previously been reported in synthetic foils and planar lipid bilayers, but our work is the first one to document the formation of nanopores and their properties in living cells. Furthermore, we have established both inhibitory and facilitatory responses of endogenous ion channels after nsEP treatment, as well as cytophysiological changes due to the osmotic imbalance. This Research Application is designed to explore the phenomenon of nanoelectroporation in living cells and to evaluate potential applications of this novel technique in research and medicine. The proposed study consists of four Specific Aims intended to characterize and improve the nanoelectroporation procedure; to reveal mechanisms that allow nanopores to perform their complex activities; and to elucidate mechanisms that underlie nsEP effects on plasma membrane barrier function and ion traffic: Specific Aim 1: Explore the dependence of nanopore formation on the physical parameters of electric pulses, optimize nanoelectroporation procedures and nanopore detection techniques. Specific Aim 2: Analyze structural and functional properties of nanopores (pore lifetime, opening diameter, ion selectivity, voltage and current sensitivity) and reveal mechanisms responsible for these properties. Specific Aim 3: Explore the impact of nanoelectroporation on the function of classic voltage-gated ion channels, and on the excitation and action potential propagation in nerve and muscle cells. Specific Aim 4: Explore mechanisms underlying nanoporation effect on plasma membrane water permeability and cell volume control. PUBLIC HEALTH RELEVANCE: This study will be focused on the new phenomenon of nanoelectroporation, which is the formation of stable, voltage- and current-sensitive, nanometer-diameter membrane pores in living cells exposed to nanosecond- duration, high-voltage electric pulses (nsEP). We will focus on physico-chemical and physiological mechanisms that underlie and determine plasma membrane nanoelectroporation and nsEP effects on endogenous ion channels and water metabolism. Anticipated results will promote the development of new medical and research applications using nsEP for deliberate modification of cell functions, particularly in nerve and muscle tissues.